Deletion of the Major Proteolytic Site of the Helicobacter pylori Cytotoxin Does Not Influence Toxin Activity but Favors Assembly of the Toxin into Hexameric Structures

The Helicobacter pylori cytotoxin is proteolytically cleaved at a flexible hydrophilic loop into two subunits. Deletion of the loop sequences had no effect on biological activity of the toxin in the HeLa cell vacuolation assay but favored the organization of the protein into hexameric rather than heptameric structures.     

Image-directed percutaneous biopsies with a biopsy gun

Core tissue for histologic study is believed by many pathologists to be more diagnostic than material from needle aspiration. Recently, a biopsy "gun" has been introduced, which simplifies core biopsies. With this device, 182 biopsies of multiple anatomic sites were performed with ultrasonic, computed tomographic, and fluoroscopic guidance and 18-gauge needles. High-quality histopathologic specimens were obtained in 177 of the biopsies, and diagnostic target tissue was obtained in 167. Only three significant complications occurred: one bleeding complication that required transfusion and two cases of pneumothorax that necessitated placement of chest tubes. The biopsy gun eliminated the disjointed movements of conventional "skinny" needle biopsies, and none of the samples demonstrated significant "crush" artifact or obscuring blood, problems that are commonly associated with manual biopsy techniques. Patient discomfort was decreased with this system compared with that of manual biopsies, and the total procedure time was reduced. Because of these distinct advantages, the authors now use the biopsy gun exclusively for all percutaneous biopsies and recommend that other institutions consider the use of this biopsy method.     

新生期大白鼠皮下注射谷氨酸单钠对成年后下丘脑α-促黑素细胞激素神经元免疫反应性的影响

本文用免疫组化方法研究了新生期大白鼠注射谷氨酸单钠(MSG)对成年后下丘脑α-促黑素细胞激素(α-MSH)免疫反应神经元的影响,结果显示MSG处理以后下丘脑弓状核区α-MSII免疫反应神经元减少甚至完全消失,但不影响下丘脑背外侧区的α-MSH神经元群。文中还讨论了这两群α-MSH神经元的生理作用。     

Linkage of the MHC to familial multiple sclerosis suggests genetic heterogeneity. The Multiple Sclerosis Genetics Group

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system. While its etiology is not well understood, genetic factors are clearly involved. Until recently, most genetic studies in MS have been association studies using the case-control design testing specific candidate genes and studying only sporadic cases. The only consistently replicated finding has been an association with the HLA-DR2 allele within the major histocompatibility complex (MHC) on chromosome 6. Using the genetic linkage design, however, evidence for and against linkage of the MHC to MS has been found, fostering suggestions that sporadic and familial MS have different etiologies. Most recently, two of four genomic screens demonstrated linkage to the MHC, although specific allelic associations were not tested. Here, a dataset of 98 multiplex families was studied to test for an association to the HLA-DR2 allele in familial MS and to determine if genetic linkage to the MHC was due solely to such an association. Three highly polymorphic markers (HLA-DR, D6S273 and TNFbeta) in the MHC demonstrated strong genetic linkage (parametric lod scores of 4.60, 2.20 and 1.24, respectively) and a specific association with the HLA-DR2 allele was confirmed (TDT; P < 0.001). Stratifying the results by HLA-DR2 status showed that the linkage results were limited to families segregating HLA-DR2 alleles. These results demonstrate that genetic linkage to the MHC can be explained by the HLA-DR2 allelic association. They also indicate that sporadic and familial MS share a common genetic susceptibility. In addition, preliminary calculations suggest that the MHC explains between 17 and 62% of the genetic etiology of MS. This heterogeneity is also supported by the minority of families showing no linkage or association with loci within the MHC.      

CTLA-4 is required for the induction of high dose oral tolerance

Mucosal and systemic administrations of high dose antigens induce long- lasting peripheral T cell tolerance. We and others have shown that high dose peripheral T cell tolerance is mediated by anergy or deletion and is preceded by T cell activation. Co-stimulatory molecules B7-1 (CD80)/B7-2 (CD86) and their counter-receptors CD28/CTLA-4 play pivotal roles in T cell activation and immune regulation. In the present study, we examined the roles of the B7 co-stimulation pathway in the generation of high dose peripheral T cell tolerance. We found that blocking B7:CD28/CTLA-4 interaction at the time of tolerance induction partially prevented T cell tolerance, whereas selective blockade of B7:CTLA-4 interaction completely abrogated peripheral T cell tolerance induced by either oral or i.p. antigens. These results suggest that CTLA-4-mediated feedback regulation plays a crucial role in the induction of high dose peripheral T cell tolerance.      

Screening for proteins with polyglutamine expansions in autosomal dominant cerebellar ataxias

Expansion of trinucleotide CAG repeats coding for polyglutamine has been implicated in five neurodegenerative disorders, including spinocerebellar ataxia (SCA) 1 and SCA3 or Machado-Joseph disease (SCA3/MJD), two forms of type I autosomal dominant cerebellar ataxias (ADCA). Using the 1C2 antibody which specifically recognizes large polyglutamine tracts, particularly those that are expanded, we recently reported the detection of proteins with pathological glutamine expansions in lymphoblasts from another form of ADCA type I, SCA2, as well as from patients presenting with the distinct phenotype of ADCA type II. We now have screened a large series of patients with ADCA or isolated cases with cerebellar ataxia, for the presence of proteins with polyglutamine expansions. A 150 kDa SCA2 protein was detected in 16 out of 40 families with ADCA type I. This corresponds to 24% of all ADCA type I families, which is much more frequent than SCA1 in this series of patients (13%). The signal intensity of the SCA2 protein was negatively correlated to age at onset, as expected for an expanded and unstable trinucleotide repeat mutation. The disease segregated with markers closely linked to the SCA2 locus in all identified SCA2 families. In addition, a specific 130 kDa protein, which segregated with the disease, was detected in lymphoblasts of patients from nine families with ADCA type II. It was also visualized in the cerebral cortex of one of the patients, demonstrating its translation in the nervous system. Finally, no new disease-related proteins containing expanded polyglutamine tracts could be detected in lymphoblasts from the remaining patients with ADCA or isolated cases with cerebellar ataxia.      

P55, an immunogenic but nonprotective 55-kilodalton Borrelia burgdorferi protein in murine Lyme disease.

Immunization of C3H mice with P55 (previously called S1), a 55-kDa Borrelia burgdorferi antigen that is immunogenic after infection, elicited a strong antibody response but did not protect mice against B. burgdorferi challenge. Mice immunized with a P55 fusion protein in complete Freund's adjuvant developed anti-P55 antibodies, detectable at a titer of 1:10,000 by immunoblotting. To determine, if a protective response had been elicited, P55-vaccinated mice were fed upon by ticks infected with B. burgdorferi. The frequency of B. burgdorferi infection was similar in P55-immunized and control mice, and spirochetes were not destroyed within ticks that fed on P55-vaccinated mice. P55 is an immunogenic antigen that does not induce a protective response in the vertebrate or invertebrate host.     

Standardization of medium for culturing Lyme disease spirochetes.

To standardize the procedure for isolating and culturing Lyme disease spirochetes, we modified the composition of the medium generally used for this purpose (BSK-II) and developed a system for its distribution. This medium contains no gelatin or agarose, and various components are used in proportions that differ from those in BSK-II. Each of the major proteinacious components was screened by substitution in samples of the complete product. The final medium was evaluated for the capacity to grow related spirochetes including Borrelia burgdorferi N40, Guilford, and JD-1 as well as strains of Borrelia hermsii (HS-1) and of Borrelia coriaceae (CO53). Each isolate developed from inocula containing as few as one to five organisms. Doubling time of B. burgdorferi during log-phase growth at 37 degrees C was 10 to 12 h. Lyme disease spirochetes were isolated in this medium from ear punch biopsies and dermal aspirates from naturally infected mice and rabbits, from dermal biopsies from a human patient, and by sampling field-collected deer ticks (Ixodes dammini). Cultured spirochetes remained infective to mice and to ticks. The medium can be stored at -20 degrees C or lower temperatures for at least 8 months without effect on its ability to support growth of small inocula to densities exceeding 10(8) spirochetes per ml. Lyme disease spirochetes remained infective to mice after being stored at -80 degrees C in this medium for at least 8 months. We anticipate that the availability of this standardized medium (Sigma Chemical Co.), supplemented with prescreened rabbit serum, will facilitate comparison of research results between laboratories and may eventually permit definitive clinical diagnosis of Lyme disease based on demonstration of the pathogen. The standardized medium is designated BSK-H.     

Canal-otolith interactions in the squirrel monkey vestibulo-ocular reflex and the influence of fixation distance

Natural head movements include angular and linear components of motion. Two classes of vestibulo-ocular reflex (VOR), mediated by the semicircular canals and otoliths (the angular and linear VOR, or AVOR and LVOR, respectively), compensate for head movements and help maintain binocular fixation on targets in space. In this study, AVOR/LVOR interactions were quantified during complex head motion over a broad range of fixation distances at a fixed stimulus frequency of 4.0 Hz. Binocular eye movements were recorded (search-coil technique) in squirrel monkeys while fixation distance (assessed by vergence) was varied using brief presentations of earth-fixed targets at various distances. Stimuli consisted of rotations around an earth-vertical axis and therefore always activated the AVOR. Horizontal and vertical AVORs were assessed when the head was centered over the axis of rotation and oriented upright (UP) and right-side-down (RD), respectively. AVOR gains increased slightly with increasing vergence in darkness, as expected given the small anterior position of the eyes in the head. Combined AVOR/LVOR responses were recorded when subjects were displaced eccentrically from the rotation axis. Eccentric rotations activated the AVOR just as when the head was centered, but added a translational stimulus which generated an LVOR component in response to interaural (IA) or dorsoventral (DV) tangential accelerations, depending on whether the head was UP or RD, respectively. When the head was eccentric and facing nose-out, the AVOR and LVOR produced ocular responses in the same plane and direction (coplanar and synergistic), and response magnitudes increased with increasing vergence. With the head facing nose-in, AVOR and LVOR response components were oppositely directed (coplanar and antagonistic). The AVOR dominated the response when fixation distance was far, and phase was compensatory for head rotation. As fixation distance decreased toward the rotation axis, responses declined to near zero, and when fixation distance approached even closer, the LVOR component dominated and response phase inverted. The same pattern was observed for both horizontal (head UP) and vertical (head RD) responses. The LVOR was recorded directly by rotating subjects eccentrically but in the nose-up (NU) orientation. The AVOR then generated torsional responses to head roll, coexistent with either horizontal or vertical LVOR responses to tangential acceleration when the subject was oriented head-out or right-side-out, respectively. Only the LVOR response components were modulated by vergence. A vectorial analysis of AVOR, LVOR, and combined responses supports the conclusion that AVOR and LVOR response components combine linearly during complex head motion. Received: 27 February 1997 / Accepted: 18 June 1997     

Partial destruction of Borrelia burgdorferi within ticks that engorged on OspE- or OspF-immunized mice.

We determined whether Borrelia burgdorferi outer surface proteins (Osps) E and F could elicit immune responses useful for a Lyme disease vaccine. Thirty days after challenge with B. burgdorferi, mice produced antibodies to OspE but not OspF, whereas antibodies to OspF were present in sera of mice obtained 90 days after infection. Examination of sera from patients with Lyme disease revealed antibodies to OspF in a small number (14%) of early-stage disease patients but in a majority (58%) of patients with late-stage disease, while antibodies to OspE were rarely detected in patients. Mice immunized with recombinant OspE or OspF produced high titers of antibodies to OspE or OspF, respectively. OspF-immunized mice were partially protected from both intradermal syringe challenge and tick-mediated transmission of B. burgdorferi while vaccination with OspE did not confer immunity. B. burgdorferi organisms were, however, substantially destroyed within ticks that engorged on either OspE- (75% reduction in the number of spirochetes within the ticks, compared with controls) or OspF (90% reduction in the number of spirochetes within the ticks)-immunized mice.